Performance of a fully-automated system on a WHO malaria microscopy evaluation slide set.

BACKGROUND: Manual microscopy remains a widely-used tool for malaria diagnosis and clinical studies, but it has inconsistent quality in the field due to variability in training and field practices. Automated diagnostic systems based on machine learning hold promise to improve quality and reproducibility of field microscopy. The World Health Organization (WHO) has designed a 55-slide set (WHO 55) for their External Competence Assessment of Malaria Microscopists (ECAMM) programme, which can also serve as a valuable benchmark for automated systems. The performance of a fully-automated malaria diagnostic system, EasyScan GO, on a WHO 55 slide set was evaluated. METHODS: The WHO 55 slide set is designed to evaluate microscopist competence in three areas of malaria diagnosis using Giemsa-stained blood films, focused on crucial field needs: malaria parasite detection, malaria parasite species identification (ID), and malaria parasite quantitation. The EasyScan GO is a fully-automated system that combines scanning of Giemsa-stained blood films with assessment algorithms to deliver malaria diagnoses. This system was tested on a WHO 55 slide set. RESULTS: The EasyScan GO achieved 94.3 % detection accuracy, 82.9 % species ID accuracy, and 50 % quantitation accuracy, corresponding to WHO microscopy competence Levels 1, 2, and 1, respectively. This is, to our knowledge, the best performance of a fully-automated system on a WHO 55 set. CONCLUSIONS: EasyScan GO's expert ratings in detection and quantitation on the WHO 55 slide set point towards its potential value in drug efficacy use-cases, as well as in some case management situations with less stringent species ID needs. Improved runtime may enable use in general case management settings.

Keep the quality high: the benefits of lot testing for the quality control of malaria rapid diagnostic tests.

BACKGROUND: The production and use of malaria rapid diagnostic tests (RDTs) has risen dramatically over the past 20 years. In view of weak or non-existing in vitro diagnostics (IVD) regulations and post-marketing surveillance (PMS) systems in malaria endemic countries, the World Health Organization, later joined by the Foundation for Innovative New Diagnostics, established an independent, centralized performance evaluation and Lot Testing (LT) programme to safeguard against poor quality of RDTs being distributed through the public health sector of malaria endemic countries. RDT performances and manufacturer quality management systems have evolved over the past decade raising questions about the future need for a centralized LT programme. RESULTS: Between 2007 and 2017, 6056 lots have been evaluated, representing approximately 1.6 Billion RDTs. A total of 69 lots (1.1%) failed the quality control. Of these failures, 26 were detected at receipt of the RDT lot in the LT laboratory, representing an estimated 7.9 million poor quality RDTs, and LT requesters were advised that RDTs were not of sufficient quality for use in patient management. Forty-three were detected after long-term storage in the laboratory, of which 24 (56%) were found to be due to a major issue with insufficient buffer volume in single use buffer vials, others predominantly showing loss of sensitivity. The annual cost of running the programme, based on expenses recorded in years 2014-2016, an estimated volume of 700 lots per year and including replenishment of quality control samples, was estimated at US$ 178,500 ($US 255 per lot tested). CONCLUSIONS: Despite the clear benefits of the centralized LT programme and its low cost compared with the potential costs of each country establishing its own PMS system for RDTs, funding concerns have made its future beyond 2020 uncertain. In order to manage the risks of misdiagnosis due to low quality RDTs, and to ensure the continued safety and reliability of malaria case management, there is a need to ensure that an effective and implementable approach to RDT quality control continues to be available to programmes in endemic countries.

A review of the WHO malaria rapid diagnostic test product testing programme (2008-2018): performance, procurement and policy.

Malaria rapid diagnostic tests (RDTs) emerged in the early 1990s into largely unregulated markets, and uncertain field performance was a major concern for the acceptance of tests for malaria case management. This, combined with the need to guide procurement decisions of UN agencies and WHO Member States, led to the creation of an independent, internationally coordinated RDT evaluation programme aiming to provide comparative performance data of commercially available RDTs. Products were assessed against Plasmodium falciparum and Plasmodium vivax samples diluted to two densities, along with malaria-negative samples from healthy individuals, and from people with immunological abnormalities or non-malarial infections. Three measures were established as indicators of performance, (i) panel detection score (PDS) determined against low density panels prepared from P. falciparum and P. vivax wild-type samples, (ii) false positive rate, and (iii) invalid rate, and minimum criteria defined. Over eight rounds of the programme, 332 products were tested. Between Rounds 1 and 8, substantial improvements were seen in all performance measures. The number of products meeting all criteria increased from 26.8% (11/41) in Round 1, to 79.4% (27/34) in Round 8. While products submitted to further evaluation rounds under compulsory re-testing did not show improvement, those voluntarily resubmitted showed significant increases in P. falciparum (p = 0.002) and P. vivax PDS (p < 0.001), with more products meeting the criteria upon re-testing. Through this programme, the differentiation of products based on comparative performance, combined with policy changes has been influential in the acceptance of malaria RDTs as a case-management tool, enabling a policy of parasite-based diagnosis prior to treatment. Publication of product testing results has produced a transparent market allowing users and procurers to clearly identify appropriate products for their situation, and could form a model for introduction of other, broad-scale diagnostics.

Use of mobile technology-based participatory mapping approaches to geolocate health facility attendees for disease surveillance in low resource settings.

BACKGROUND: Identifying fine-scale spatial patterns of disease is essential for effective disease control and elimination programmes. In low resource areas without formal addresses, novel strategies are needed to locate residences of individuals attending health facilities in order to efficiently map disease patterns. We aimed to assess the use of Android tablet-based applications containing high resolution maps to geolocate individual residences, whilst comparing the functionality, usability and cost of three software packages designed to collect spatial information. RESULTS: Using Open Data Kit GeoODK, we designed and piloted an electronic questionnaire for rolling cross sectional surveys of health facility attendees as part of a malaria elimination campaign in two predominantly rural sites in the Rizal, Palawan, the Philippines and Kulon Progo Regency, Yogyakarta, Indonesia. The majority of health workers were able to use the tablets effectively, including locating participant households on electronic maps. For all households sampled (n = 603), health facility workers were able to retrospectively find the participant household using the Global Positioning System (GPS) coordinates and data collected by tablet computers. Median distance between actual house locations and points collected on the tablet was 116 m (IQR 42-368) in Rizal and 493 m (IQR 258-886) in Kulon Progo Regency. Accuracy varied between health facilities and decreased in less populated areas with fewer prominent landmarks. CONCLUSIONS: Results demonstrate the utility of this approach to develop real-time high-resolution maps of disease in resource-poor environments. This method provides an attractive approach for quickly obtaining spatial information on individuals presenting at health facilities in resource poor areas where formal addresses are unavailable and internet connectivity is limited. Further research is needed on how to integrate these with other health data management systems and implement in a wider operational context.

Circulating antibodies against Plasmodium falciparum histidine-rich proteins 2 interfere with antigen detection by rapid diagnostic tests.

BACKGROUND: Rapid diagnostic tests (RDTs) for detection of Plasmodium falciparum infection that target P. falciparum histidine-rich protein 2 (PfHRP2), a protein that circulates in the blood of patients infected with this species of malaria, are widely used to guide case management. Understanding determinants of PfHRP2 availability in circulation is therefore essential to understanding the performance of PfHRP2-detecting RDTs. METHODS: The possibility that pre-formed host anti-PfHRP2 antibodies may block target antigen detection, thereby causing false negative test results was investigated in this study. RESULTS: Anti-PfHRP2 antibodies were detected in 19/75 (25%) of plasma samples collected from patients with acute malaria from Cambodia, Nigeria and the Philippines, as well as in 3/28 (10.7%) asymptomatic Solomon Islands residents. Pre-incubation of plasma samples from subjects with high-titre anti-PfHRP2 antibodies with soluble PfHRP2 blocked the detection of the target antigen on two of the three brands of RDTs tested, leading to false negative results. Pre-incubation of the plasma with intact parasitized erythrocytes resulted in a reduction of band intensity at the highest parasite density, and a reduction of lower detection threshold by ten-fold on all three brands of RDTs tested. CONCLUSIONS: These observations indicate possible reduced sensitivity for diagnosis of P. falciparum malaria using PfHRP2-detecting RDTs among people with high levels of specific antibodies and low density infection, as well as possible interference with tests configured to detect soluble PfHRP2 in saliva or urine samples. Further investigations are required to assess the impact of pre-formed anti-PfHRP2 antibodies on RDT performance in different transmission settings.

Analytical approaches for antimalarial antibody responses to confirm historical and recent malaria transmission: an example from the Philippines.

Background: Assessing the status of malaria transmission in endemic areas becomes increasingly challenging as countries approach elimination. Serology can provide robust estimates of malaria transmission intensities, and multiplex serological assays allow for simultaneous assessment of markers of recent and historical malaria exposure. Methods: Here, we evaluated different statistical and machine learning methods for analyzing multiplex malaria-specific antibody response data to classify recent and historical exposure to Plasmodium falciparum and Plasmodium vivax. To assess these methods, we utilized samples from a health-facility based survey (n = 9132) in the Philippines, where we quantified antibody responses against 8 P. falciparum and 6 P. vivax-specific antigens from 3 sites with varying transmission intensity. Findings: Measurements of antibody responses and seroprevalence were consistent with the 3 sites' known endemicity status. Among the models tested, a machine learning (ML) approach (Random Forest model) using 4 serological markers (PfGLURP R2, Etramp5.Ag1, GEXP18, and PfMSP119) gave better predictions for P. falciparum recent infection in Palawan (AUC: 0.9591, CI 0.9497-0.9684) than individual antigen seropositivity. Although the ML approach did not improve P. vivax infection predictions, ML classifications confirmed the absence of recent exposure to P. falciparum and P. vivax in both Occidental Mindoro and Bataan. For predicting historical P. falciparum and P. vivax transmission, seroprevalence and seroconversion rates based on cumulative exposure markers AMA1 and MSP119 showed reliable trends in the 3 sites. Interpretation: Our study emphasizes the utility of serological markers in predicting recent and historical exposure in a sub-national elimination setting, and also highlights the potential use of machine learning models using multiplex antibody responses to improve assessment of the malaria transmission status of countries aiming for elimination. This work also provides baseline antibody data for monitoring risk in malaria-endemic areas in the Philippines. Funding: Newton Fund, Philippine Council for Health Research and Development, UK Medical Research Council.

Developing standards for malaria microscopy: external competency assessment for malaria microscopists in the Asia-Pacific.

BACKGROUND: Malaria diagnosis has received renewed interest in recent years, associated with the increasing accessibility of accurate diagnosis through the introduction of rapid diagnostic tests and new World Health Organization guidelines recommending parasite-based diagnosis prior to anti-malarial therapy. However, light microscopy, established over 100 years ago and frequently considered the reference standard for clinical diagnosis, has been neglected in control programmes and in the malaria literature and evidence suggests field standards are commonly poor. Microscopy remains the most accessible method for parasite quantitation, for drug efficacy monitoring, and as a reference of assessing other diagnostic tools. This mismatch between quality and need highlights the importance of the establishment of reliable standards and procedures for assessing and assuring quality. This paper describes the development, function and impact of a multi-country microscopy external quality assurance network set up for this purpose in Asia. METHODS: Surveys were used for key informants and past participants for feedback on the quality assurance programme. Competency scores for each country from 14 participating countries were compiled for analyses using paired sample t-tests. In-depth interviews were conducted with key informants including the programme facilitators and national level microscopists. RESULTS: External assessments and limited retraining through a formalized programme based on a reference slide bank has demonstrated an increase in standards of competence of senior microscopists over a relatively short period of time, at a potentially sustainable cost. The network involved in the programme now exceeds 14 countries in the Asia-Pacific, and the methods are extended to other regions. CONCLUSIONS: While the impact on national programmes varies, it has translated in some instances into a strengthening of national microscopy standards and offers a possibility both for supporting revival of national microcopy programmes, and for the development of globally recognized standards of competency needed both for patient management and field research.

Malaria rapid diagnostic test transport and storage conditions in Burkina Faso, Senegal, Ethiopia and the Philippines.

BACKGROUND: As more point of care diagnostics become available, the need to transport and store perishable medical commodities to remote locations increases. As with other diagnostics, malaria rapid diagnostic tests (RDTs) must be highly reliable at point of use, but exposure to adverse environmental conditions during distribution has the potential to degrade tests and accuracy. In remote locations, poor quality diagnostics and drugs may have significant negative health impact that is not readily detectable by routine monitoring. This study assessed temperature and humidity throughout supply chains used to transport and store health commodities, such as RDTs. METHODS: Monitoring devices capable of recording temperature and humidity were deployed to Burkina Faso (8), Senegal (10), Ethiopia (13) and the Philippines (6) over a 13-month period. The devices travelled through government supply chains, usually alongside RDTs, to health facilities where RDTs are stored, distributed and used. The recording period spanned just over a year, in order to avoid any biases related to seasonal temperature variations. RESULTS: In the four countries, storage and transport temperatures regularly exceeded 30.0°C; maximum humidity level recorded was above 94% for the four countries. In three of the four countries, temperatures recorded at central storage facilities exceeded pharmaceutical storage standards for over 20% of the time, in another case for a majority of the time; and sometimes exceeded storage temperatures at peripheral sites. CONCLUSIONS: Malaria RDTs were regularly exposed to temperatures above recommended limits for many commercially-available RDTs and other medical commodities such as drugs, but rarely exceeded the recommended storage limits for particular products in use in these countries. The results underline the need to select RDTs, and other commodities, according to expected field conditions, actively manage the environmental conditions in supply chains in tropical and sub-tropical climates. This would benefit from a re-visit of current global standards on stability of medical commodities based in tropical and sub-tropical climatic zones.

Prevalence and temporal changes of mutations linked to antimalarial drug resistance in Plasmodium falciparum and Plasmodium vivax in Palawan, Philippines.

OBJECTIVE: This study provides 2016 data on the prevalence of key single nucleotide polymorphisms (SNPs) associated with antimalarial drug resistance in Palawan, Philippines. Findings were combined with historical data to model temporal changes in the prevalence of these SNPs in Plasmodium isolates. METHODS: Plasmodium isolates were genotyped using drug resistance markers pfmdr1, pfcrt, pfdhfr, pfdhps, kelch-13, pvmdr1, pvdhfr, and pvdhps. Temporal trends in the probability of mutations were estimated as a function of time using a binomial generalised linear model. RESULTS: All samples sequenced for Plasmodium falciparum chloroquine markers pfmdr1 and pfcrt had wild-type alleles. Varying mutation patterns were observed for the sulphadoxine/pyrimethamine markers pfdhps and pfdhfr; complete quintuplet mutations were not found. No SNPs were observed for the artemisinin marker kelch-13. For Plasmodium vivax, differing patterns were detected for pvmdr1, pvdhfr, and pvdhps. CONCLUSIONS: The study findings suggest that the current drugs remain effective and that there is limited importation and establishment of resistant parasites in the area. Clear temporal trends were recognised, with prominent decreases in the proportions of pfcrt and pfmdr mutations detected within the past 15 years, consistent with a change in antimalarial drug policy. Continuous surveillance of antimalarial drug resistance is important to support malaria elimination efforts.

Laboratory demonstration of a prozone-like effect in HRP2-detecting malaria rapid diagnostic tests: implications for clinical management.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are now widely used for prompt on-site diagnosis in remote endemic areas where reliable microscopy is absent. Aberrant results, whereby negative test results occur at high parasite densities, have been variously reported for over a decade and have led to questions regarding the reliability of the tests in clinical use. METHODS: In the first trial, serial dilutions of recombinant HRP2 antigen were tested on an HRP2-detectiing RDT. In a second trial, serial dilutions of culture-derived Plasmodium falciparum parasites were tested against three HRP2-detecting RDTs. RESULTS: A prozone-like effect occurred in RDTs at a high concentration of the target antigen, histidine-rich protein-2 (above 15,000 ng/ml), a level that corresponds to more than 312000 parasites per µL. Similar results were noted on three RDT products using dilutions of cultured parasites up to a parasite density of 25%. While reduced line intensity was observed, no false negative results occurred. CONCLUSIONS: These results suggest that false-negative malaria RDT results will rarely occur due to a prozone-like effect in high-density infections, and other causes are more likely. However, RDT line intensity is poorly indicative of parasite density in high-density infections and RDTs should, therefore, not be considered quantitative. Immediate management of suspected severe malaria should rely on clinical assessment or microscopy. Evaluation against high concentrations of antigen should be considered in malaria RDT product development and lot-release testing, to ensure that very weak or false negative results will not occur at antigen concentrations that might be seen clinically.

Blood transfer devices for malaria rapid diagnostic tests: evaluation of accuracy, safety and ease of use.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are increasingly used by remote health personnel with minimal training in laboratory techniques. RDTs must, therefore, be as simple, safe and reliable as possible. Transfer of blood from the patient to the RDT is critical to safety and accuracy, and poses a significant challenge to many users. Blood transfer devices were evaluated for accuracy and precision of volume transferred, safety and ease of use, to identify the most appropriate devices for use with RDTs in routine clinical care. METHODS: Five devices, a loop, straw-pipette, calibrated pipette, glass capillary tube, and a new inverted cup device, were evaluated in Nigeria, the Philippines and Uganda. The 227 participating health workers used each device to transfer blood from a simulated finger-prick site to filter paper. For each transfer, the number of attempts required to collect and deposit blood and any spilling of blood during transfer were recorded. Perceptions of ease of use and safety of each device were recorded for each participant. Blood volume transferred was calculated from the area of blood spots deposited on filter paper. RESULTS: The overall mean volumes transferred by devices differed significantly from the target volume of 5 microliters (p < 0.001). The inverted cup (4.6 microliters) most closely approximated the target volume. The glass capillary was excluded from volume analysis as the estimation method used is not compatible with this device. The calibrated pipette accounted for the largest proportion of blood exposures (23/225, 10%); exposures ranged from 2% to 6% for the other four devices. The inverted cup was considered easiest to use in blood collection (206/226, 91%); the straw-pipette and calibrated pipette were rated lowest (143/225 [64%] and 135/225 [60%] respectively). Overall, the inverted cup was the most preferred device (72%, 163/227), followed by the loop (61%, 138/227). CONCLUSIONS: The performance of blood transfer devices varied in this evaluation of accuracy, blood safety, ease of use, and user preference. The inverted cup design achieved the highest overall performance, while the loop also performed well. These findings have relevance for any point-of-care diagnostics that require blood sampling.

Enhanced Health Facility Surveys to Support Malaria Control and Elimination across Different Transmission Settings in the Philippines.

Following substantial progress in malaria control in the Philippines, new surveillance approaches are needed to identify and target residual malaria transmission. This study evaluated an enhanced surveillance approach using rolling cross-sectional surveys of all health facility attendees augmented with molecular diagnostics and geolocation. Facility surveys were carried out in three sites representing different transmission intensities: Morong, Bataan (pre-elimination), Abra de Ilog, Occidental Mindoro (stable medium risk), and Rizal, Palawan (high risk, control). Only one rapid diagnostic test (RDT)-positive infection and no PCR confirmed infections were found in Bataan and Occidental Mindoro, suggesting the absence of transmission. In Palawan, the inclusion of all health facility attendees, regardless of symptoms, and use of molecular diagnostics identified 313 infected individuals in addition to 300 cases identified by routine screening of febrile patients with the RDT or microscopy. Of these, the majority (313/613) were subpatent infections and only detected using molecular methods. Simultaneous collection of GPS coordinates on tablet-based applications allowed real-time mapping of malaria infections. Risk factor analysis showed higher risks in children and indigenous groups, with bed net use having a protective effect. Subpatent infections were more common in men and older age-groups. Overall, malaria risks were not associated with participants' classification, and some of the non-patient clinic attendees reported febrile illnesses (1.9%, 26/1,369), despite not seeking treatment, highlighting the widespread distribution of infection in communities. Together, these data illustrate the utility of health facility-based surveys to augment surveillance data to increase the probability of detecting infections in the wider community.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests.

BACKGROUND: Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. METHODS: The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. RESULTS: Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. CONCLUSIONS: The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.

Comparison of Commercial ELISA Kits to Confirm the Absence of Transmission in Malaria Elimination Settings.

Background: Antimalarial antibody measurements are useful because they reflect historical and recent exposure to malaria. As such, they may provide additional information to assess ongoing transmission in low endemic or pre-elimination settings where cases are rare. In addition, the absence of antibody responses in certain individuals can indicate the cessation of transmission. Commercial malaria enzyme-linked immunosorbent assays (ELISA) detect antimalarial antibodies and are commonly used to screen blood donations for possible malaria infection. However, there is no standardized test to detect antimalarial antibodies for epidemiological use. Here we compared five commercially available ELISA kits (Trinity Biotech, newbio, DiaPro, Cellabs, and NovaTec) in search of a standardized tool for supporting claims of absence of malaria transmission. For comparison, a research-based (RB) ELISA protocol was performed alongside the commercial kits. Results: The commercial kits were first compared using serum samples from known malaria-unexposed individuals (n = 223) and Toxoplasma-infected individuals (n = 191) to assess specificity and cross-reactivity against non-malaria infections. In addition, 134 samples from ≥10-year-olds collected in a hyperendemic region in the Gambia in the early 1990s were used to assess sensitivity. Three out of five kits showed high sensitivity (90-92%), high specificity (98-99%), low cross-reactivity (0-3%) and were considered user-friendly (Trinity Biotech, newbio and NovaTec). Two of these kits (Trinity Biotech and NovaTec) were taken forward for epidemiological evaluation and results were compared to those using the RB-ELISA. Samples from two pre-elimination settings (Praia, Cape Verde; n = 1,396, and Bataan, the Philippines; n = 1,824) were tested. Serological results from both the Trinity Biotech kit and the RB-ELISA concurred with recent passively detected case counts in both settings. Results from the Trinity Biotech kit reflected a significant decrease in the number of reported cases in Bataan in the 1990s better than the RB-ELISA. Results from the NovaTec kit did not reflect transmission patterns in either setting. Conclusion: The Trinity Biotech commercial ELISA kit was considered reliable for epidemiological use and accurately described transmission patterns in two (previously) malaria endemic settings. The use of this simple and standardized serological tool may aid national control and elimination programs by confirming that regions are free from malaria.

Inter-rater reliability of malaria parasite counts and comparison of methods.

BACKGROUND: The introduction of artemesinin-based treatment for falciparum malaria has led to a shift away from symptom-based diagnosis. Diagnosis may be achieved by using rapid non-microscopic diagnostic tests (RDTs), of which there are many available. Light microscopy, however, has a central role in parasite identification and quantification and remains the main method of parasite-based diagnosis in clinic and hospital settings and is necessary for monitoring the accuracy of RDTs. The World Health Organization has prepared a proficiency testing panel containing a range of malaria-positive blood samples of known parasitaemia, to be used for the assessment of commercially available malaria RDTs. Different blood film and counting methods may be used for this purpose, which raises questions regarding accuracy and reproducibility. A comparison was made of the established methods for parasitaemia estimation to determine which would give the least inter-rater and inter-method variation METHODS: Experienced malaria microscopists counted asexual parasitaemia on different slides using three methods; the thin film method using the total erythrocyte count, the thick film method using the total white cell count and the Earle and Perez method. All the slides were stained using Giemsa pH 7.2. Analysis of variance (ANOVA) models were used to find the inter-rater reliability for the different methods. The paired t-test was used to assess any systematic bias between the two methods, and a regression analysis was used to see if there was a changing bias with parasite count level. RESULTS: The thin blood film gave parasite counts around 30% higher than those obtained by the thick film and Earle and Perez methods, but exhibited a loss of sensitivity with low parasitaemia. The thick film and Earle and Perez methods showed little or no bias in counts between the two methods, however, estimated inter-rater reliability was slightly better for the thick film method. CONCLUSION: The thin film method gave results closer to the true parasite count but is not feasible at a parasitaemia below 500 parasites per microlitre. The thick film method was both reproducible and practical for this project. The determination of malarial parasitaemia must be applied by skilled operators using standardized techniques.

Evaluation of three parasite lactate dehydrogenase-based rapid diagnostic tests for the diagnosis of falciparum and vivax malaria.

BACKGROUND: In areas where non-falciparum malaria is common rapid diagnostic tests (RDTs) capable of distinguishing malaria species reliably are needed. Such tests are often based on the detection of parasite lactate dehydrogenase (pLDH). METHODS: In Dawei, southern Myanmar, three pLDH based RDTs (CareStart Malaria pLDH (Pan), CareStart Malaria pLDH (Pan, Pf) and OptiMAL-IT)were evaluated in patients presenting with clinically suspected malaria. Each RDT was read independently by two readers. A subset of patients with microscopically confirmed malaria had their RDTs repeated on days 2, 7 and then weekly until negative. At the end of the study, samples of study batches were sent for heat stability testing. RESULTS: Between August and November 2007, 1004 patients aged between 1 and 93 years were enrolled in the study. Slide microscopy (the reference standard) diagnosed 213 Plasmodium vivax (Pv) monoinfections, 98 Plasmodium falciparum (Pf) mono-infections and no malaria in 650 cases. The sensitivities (sens) and specificities (spec), of the RDTs for the detection of malaria were- CareStart Malaria pLDH (Pan) test: sens 89.1% [CI95 84.2-92.6], spec 97.6% [CI95 96.5-98.4]. OptiMal-IT: Pf+/- other species detection: sens 95.2% [CI95 87.5-98.2], spec 94.7% [CI95 93.3-95.8]; non-Pf detection alone: sens 89.6% [CI95 83.6-93.6], spec 96.5% [CI95 94.8-97.7]. CareStart Malaria pLDH (Pan, Pf): Pf+/- other species: sens 93.5% [CI95 85.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI95 71.1-84.4], spec 97.8% [CI95 96.3-98.7]. Inter-observer agreement was excellent for all tests (kappa > 0.9). The median time for the RDTs to become negative was two days for the CareStart Malaria tests and seven days for OptiMAL-IT. Tests were heat stable up to 90 days except for OptiMAL-IT (Pf specific pLDH stable to day 20 at 35 degrees C). CONCLUSION: None of the pLDH-based RDTs evaluated was able to detect non-falciparum malaria with high sensitivity, particularly at low parasitaemias. OptiMAL-IT performed best overall and would perform best in an area of high malaria prevalence among screened fever cases. However, heat stability was unacceptable and the number of steps to perform this test is a significant drawback in the field. A reliable, heat-stable, highly sensitive RDT, capable of diagnosing all Plasmodium species has yet to be identified.

Human Infections with Plasmodium knowlesi, the Philippines.

Five human cases of infection with the simian malaria parasite Plasmodium knowlesi from Palawan, the Philippines, were confirmed by nested PCR. This study suggests that this zoonotic infection is found across a relatively wide area in Palawan and documents autochthonous cases in the country.

An assessment of various blood collection and transfer methods used for malaria rapid diagnostic tests.

BACKGROUND: Four blood collection and transfer devices commonly used for malaria rapid diagnostic tests (RDTs) were assessed for their consistency, accuracy and ease of use in the hands of laboratory technicians and village health workers. METHODS: Laboratory technicians and village health workers collected blood from a finger prick using each device in random order, and deposited the blood either on filter paper or into a suitable casette-type RDT. Consistency and accuracy of volume delivered was determined by comparing the measurements of the resulting blood spots/heights with the measurements of laboratory-prepared pipetted standard volumes. The effect of varying blood volumes on RDT sensitivity and ease of use was also observed. RESULTS: There was high variability in blood volume collected by the devices, with the straw and the loop, the most preferred devices, usually transferring volumes greater than intended, while the glass capillary tube and the plastic pipette transferring less volume than intended or none at all. Varying the blood volume delivered to RDTs indicated that this variation is critical to RDT sensitivity only when the transferred volume is very low. CONCLUSION: None of the blood transfer devices assessed performed consistently well. Adequate training on their use is clearly necessary, with more development efforts for improved designs to be used by remote health workers, in mind.

Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests.

Rising costs of antimalarial agents are increasing the demand for accurate diagnosis of malaria. Rapid diagnostic tests (RDTs) offer great potential to improve the diagnosis of malaria, particularly in remote areas. Many RDTs are based on the detection of Plasmodium falciparum histidine-rich protein (PfHRP) 2, but reports from field tests have questioned their sensitivity and reliability. We hypothesize that the variability in the results of PfHRP2-based RDTs is related to the variability in the target antigen. We tested this hypothesis by examining the genetic diversity of PfHRP2, which includes numerous amino acid repeats, in 75 P. falciparum lines and isolates originating from 19 countries and testing a subset of parasites by use of 2 PfHRP2-based RDTs. We observed extensive diversity in PfHRP2 sequences, both within and between countries. Logistic regression analysis indicated that 2 types of repeats were predictive of RDT detection sensitivity (87.5% accuracy), with predictions suggesting that only 84% of P. falciparum parasites in the Asia-Pacific region are likely to be detected at densities < or = 250 parasites/microL. Our data also indicated that PfHRP3 may play a role in the performance of PfHRP2-based RDTs. These findings provide an alternative explanation for the variable sensitivity in field tests of malaria RDTs that is not due to the quality of the RDTs.